Regulation of AU-rich element-mediated mRNA decay

富含 AU 元件介导的 mRNA 衰减的调节

• 批准号: 7884873
• 负责人: CHING-YI CHEN
• 金额: $ 30.77万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7884873
• 关键词: Applications Grants; Be++ element; Beryllium; Binding Proteins; Biochemical; Biological Assay; Cell Line; Cells; Cytoplasm; DDX1 gene; Defect; Development; Disease; Double-Stranded RNA; Down-Regulation; Elements; Embryo; Fibroblasts; Gene Expression; Generations; Genes; Goals; Grant; Herpesvirus 1; Immune; Immune System Diseases; Immune response; Immunoprecipitation; Indium; Infection; Inflammation; Inflammatory; Interferon Type I; Interferons; Knockout Mice; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Mediating; Messenger RNA; Microarray Analysis; Molecular; Mus; Natural Immunity; Pathogenesis; Pathologic Processes; Phenotype; Physiological; Play; Post-Transcriptional Regulation; Procedures; Process; Proteins; RNA Decay; RNA Degradation; RNA Helicase; RNA Interference; RNA Splicing; RNA Stability; Refractory; Regulation; Research; Ribonucleoproteins; Role; Scheme; Series; Techniques; Testing; Tissues; Untranslated Regions; Up-Regulation; Virus Diseases; Wild Type Mouse; alternative treatment; cis acting element; cytokine; established cell line; genetic regulatory protein; genome wide association study; genome-wide; genome-wide analysis; homologous recombination; in vivo; insight; mRNA Decay; macrophage; protein K; protein complex; protein function; public health relevance; research study; tool

DESCRIPTION (provided by applicant): Regulation of mRNA stability plays a critical role in the control of gene expression. Dysregulated mRNA turnover has been implicated in various pathological processes. Understanding the regulation of mRNA decay should provide insights into the pathogenesis of certain disease. An important cis-acting element responsible for rapid mRNA decay in mammalian cells is the AU-rich element (ARE), which directs mRNA decay through a process referred to as ARE-mediated mRNA decay (AMD). We have investigated the function of KSRP, a decay-promoting ARE-binding protein, in AMD for the past seven years. To further understand the regulation of KSRP activity in AMD and identify it's in vivo physiological mRNA targets, we have initiated a purification scheme to isolate proteins that associate with KSRP and have generated Ksrp- null mice. This grant renewal will utilize a series of biochemical and molecular approaches to achieve the following specific aims. Specific aim 1 is to characterize function of DDX1, an RNA helicase that was found to co-purify with KSRP, in regulation of AMD and the subcellular localization of KSRP. This aim will also purify KSRP complexes in a large scale, identify proteins that co-purify with KSRP by mass spectrometry, and investigate their roles in the regulation of AMD. Specific aim 2 is to investigate the roles of Ksrp in post- transcriptional control of type I interferon (IFN-1 and IFN-2) gene expression and in virus infection using Ksrp knockout mice. This aim will examine whether Ksrp regulates the decay of Ifna and Ifnb mRNAs and the effect of Ksrp deficiency on virus infection. Specific aim 3 is to identify Ksrp target mRNAs in LPS- stimulated macrophages. We will identify mRNAs that are stabilized in LPS-stimulated Ksrp-/- macrophages by genome-wide analysis of mRNA decay and those associated with Ksrp by ribonucleoprotein immunoprecipitation and microarray analysis. Our long-term goals are to understand the mechanism by which KSRP regulates AMD, to identify it's in vivo mRNA targets, and to characterize phenotypes associated with the dysregulation of the targets. The generation of Ksrp knockout mice should provide a valuable tool to study the post-transcriptional regulation of certain cytokine mRNAs by Ksrp, which should contribute to our understanding of the pathogenesis of inflammatory and immunological diseases resulting from a defect in mRNA decay. PUBLIC HEALTH RELEVANCE: Aberrant regulation of messenger RNA (mRNA) stability has been implicated in various pathological processes. Understanding the regulation of mRNA degradation would provide insights into the disease pathogenesis. Our research should allow for the development of alternative treatments for immune and inflammatory diseases and cancer.

描述（由申请人提供）：mRNA稳定性的调节在基因表达的控制中起关键作用。失调的mRNA周转已涉及各种病理过程。了解mRNA衰变的调节应该为某些疾病的发病机制提供见解。负责哺乳动物细胞中快速mRNA衰变的重要顺式作用元件是富含AU的元件（ARE），其通过称为ARE介导的mRNA衰变（AMD）的过程指导mRNA衰变。在过去的七年中，我们研究了KSRP（一种促进衰减的ARE结合蛋白）在AMD中的功能。为了进一步理解AMD中KSRP活性的调节并鉴定其体内生理mRNA靶标，我们已经启动了纯化方案以分离与KSRP相关的蛋白质并产生Ksrp缺失小鼠。这项资助将利用一系列生物化学和分子方法来实现以下具体目标。具体目标1是表征DDX 1（发现与KSRP共纯化的RNA解旋酶）在AMD的调节和KSRP的亚细胞定位中的功能。该目标还将大规模纯化KSRP复合物，通过质谱鉴定与KSRP共纯化的蛋白质，并研究它们在AMD调节中的作用。具体目标2是使用Ksrp敲除小鼠研究Ksrp在I型干扰素（IFN-1和IFN-2）基因表达的转录后控制和病毒感染中的作用。该目的将检查Ksrp是否调节Ifna和Ifnb mRNA的衰变以及Ksrp缺乏对病毒感染的影响。具体目的3是鉴定LPS刺激的巨噬细胞中的Ksrp靶mRNA。我们将通过全基因组mRNA衰变分析确定LPS刺激的Ksrp-/-巨噬细胞中稳定的mRNA，并通过核糖核蛋白免疫沉淀和微阵列分析确定与Ksrp相关的mRNA。我们的长期目标是了解KSRP调节AMD的机制，鉴定其体内mRNA靶点，并表征与靶点失调相关的表型。Ksrp基因敲除小鼠的产生应该提供一个有价值的工具，研究转录后的某些细胞因子mRNA的调控Ksrp，这应该有助于我们了解的炎症和免疫性疾病的发病机制所造成的mRNA衰变缺陷。 公共卫生相关性：信使RNA（mRNA）稳定性的异常调节与各种病理过程有关。了解mRNA降解的调节将有助于了解疾病的发病机制。我们的研究应该允许开发免疫和炎症性疾病和癌症的替代治疗方法。